致病性鸡大肠杆菌pilA基因和外膜蛋白C基因的克隆、表达及其免疫原性

Cloning, Expression and Immunity of pilA Gene and ompC Gene from Avian Pathogenic Escherichia coli

摘要:

根据GenBank公布的致病性鸡大肠杆菌的Ⅰ型菌毛pilA基因和外膜蛋白C基因序列, 分别设计了两对引物,并以分离的致病性鸡大肠杆菌基因组为模板, 经PCR特异性扩增出pilA基因和ompC基因, 基因产物大小为别为549 bp和1104 bp, 与GenBank报道的参考菌株的两个基因序列的同源性为高达98.18%和97.28%。将扩增得到的两个基因分别定向克隆到原核表达载体pET-28a中, 得到两个重组质粒pETpilA和pETompC, 转化大肠杆菌BL21(DE3)中, 得到重组菌株BL21(pETpilA)和BL21(pETompC), 经IPTG诱导后, SDS-PAGE分析分别可见表达的20 kD和40.9 kD的特异条带; Western blotting结果表明, 两种蛋白可与抗体发生特异性结合, 说明其具有良好的免疫原性。将表达的菌毛蛋白和外膜蛋白的菌株分别制成基因工程疫苗, 免疫小鼠后, 具有很好的保护能力, 表明这两株基因工程菌株有望作为鸡致病性大肠杆菌基因工程疫苗的候选生产菌株。

Abstract:

In order to amplify pilA gene and ompC gene of avian pathogenic Escherichia coli (APEC) strain, two pairs of primers were designed according to the GenBank sequences, and a 549 bp pilA gene and a 1104 bp ompC gene were obtained by PCR separately. Sequence analysis indicated that the homology of the nucleotide sequence of AEPC strain to those other reference strains was 98.18% of the pilA gene and 97.28% of the ompC gene. Two expression plasmids pETpilA and pETompC were constructed by inserting pilA gene and ompC gene into the prokaryotic expression vector pET-28a. The two plasmids were transformated into E. coli BL21 separately and two recombinant strains BL21 (pETpilA) and BL21 (pETompC) were obtained. The type 1 fimbraie and the out membrane protein were highly expressed when the recombinant strain BL21 (pETpilA) and BL21 (pETompC) were induced by IPTG. Two specific proteins were detected by SDS-PAGE and immunogenicity of the expressed protein was confirmed by Western blotting and ELISA. The expressed fimbraie and OmpC were transformed into vaccine. The protective immune response was proved after the mice were immunized with the two vaccines. The results showed that the recombinant strain BL21 (pETpilA) and BL21 (pETompC) could be as candidate vaccine to provide protective immune response against AEPC infection.